Griseolutein T from Streptomyces seoulensis, newly identified via combined-culture with Tsukamurella pulmonis, as an efficacious therapeutic agent against multidrug-resistant bacteria

Bacterial interactions can affect the production of secondary metabolites and, therefore, provide a promising approach to exploring new microbial compounds. In this study, we screened actinomycetes isolated from Hegura Island, Ishikawa Prefecture, Japan, to discover new antibiotics through combined-culture with Tsukamurella pulmonis TP-B0596. Three new phenazine-class antibiotics, griseoluteins T (1), C (2), and D (3), along with two known related metabolites, griseoluteic acid (4) and griseolutein A (5), were detected in both mono- and combined-cultures of Streptomyces seoulensis HEK131 with T. pulmonis at different production levels. Detailed spectroscopic analysis revealed that 1 contained a dihydrophenazine core, and was converted to 5 by accepting oxidation spontaneously. 1, containing a dihydrophenazine group, was relatively unstable under oxidative conditions, and the addition of ascorbate was required during the isolation of the compound. 2 and 3 were found to be cysteine-adducts analogous to 4, and their productivity was increased in the combined-culture. We further assessed the antibacterial activities of 1 against clinically significant Gram-positive pathogenic bacteria, including 30 methicillin-resistant Staphylococcus aureus (MRSA), 27 vancomycin-resistant Enterococci (VRE), and 17 Clostridioides difficile. Notably, 1 was found to possess higher antibacterial activity against these microorganisms than several clinically important antibiotics, while displaying lower cytotoxicity against HeLa-S3 cells.